The polymorphism of transferrin (Tf) is currently being studied by isoelectric focusing in carrier ampholyte-generated pH gradients, carrier ampholyte-separator pH gradients or in immobilized pH gradients. Details for obtaining reproducible results with each of the three procedures are outlined. The effectiveness of pretreatment of serum samples with ferrous/ferric salts is discussed, and incubation times optimized after spectrophotometric measurement of the monoferric Tf conversion. Most of the presently available commercial batches of carrier ampholytes do not reliably discriminate the six common TfC subtypes. Resolution of C1, C3 and C2 was achieved by adding 20 to 90 mM HEPES slab gels prepared with various carrier ampholytes. Isoelectric focusing in carrier ampholyte-separator pH gradients cannot be recommended as a standard typing procedure because the results strongly depend on the batch of carrier ampholytes. Tf subtype resolution was only achieved by using isoelectric focusing in immobilized pH gradients with pH slopes reliably reproducible from one experiment to another. Two major shortcomings of immobilized pH gradients are a marked tendency to protein precipitation at the application site and an interaction of proteins with the charged matrix. A protocol for Tf subtyping in immobilized pH gradients is described, based on prior desialylation of samples instead of pretreatment with iron. Sample entry into the matrix was optimized by addition of 5 mM Tris to the gels, and initially running them at low voltage. Recommendations are provided for the application of Tf typing for paternity testing.